3′‐O‐[5‐azidonaphthoyl]‐ADP has been synthesized as a photoreactive analog to 3′‐O‐naphthoyl(1)‐ADP which is known to bind to the high‐affinity nucleotide sites of mitochondrial F1‐ATPase, considered to be the catalytic sites. The photolabel in (he dark acts as a ligand to F1‐ATPase and as a competitive inhibitor with K1= 11 μM. Binding to the enzyme is accompanied by a quench of endogenous protein fluorescence leveling off at an occupancy or 1 mol/mol F1, whereas the total number of reversible sites accessible to the analog is 3 mol/mol F1 as measured by isotope studies. Covalent insertion by near ultraviolet activation of the probe yields labeling of both α and β polypeptides of F1: it is accompanied by corresponding removal of reversible high‐affinity sites for ADP or naphthoyl‐ADP and by an inhibition of the enzyme; total inactivation occurs at a covalent occupancy of 2 mol/mol F1. This is the maximum number of sites accessible to covalent modification by the label; one reversible site is still available in the totally inactivated enzyme. This observation is discussed in terms of a stochastic model requiring a minimum of two interacting catalytic domains out of three in order to commence catalysis.
|Number of pages||8|
|Journal||European Journal of Biochemistry|
|State||Published - Sep 1984|